This invention relates to a filtration apparatus and method for the separation of microscopic entities from a fluid (liquid or gas) and subsequent visual or imaging microscopic analysis of the entities separated thereon either directly or after treatment of the entities whilst on the apparatus in order to enhance their subsequent visualization and/or imaging. Such treatment can comprise reaction with reagents contained in other solutions that can be made to imbibe and/or pass through the filtration apparatus and which can be washed with solutions that can be made to imbibe and/or pass through the filtration apparatus. An example of a specific area of application is in the microbiological testing of fluids in order to detect, identify and/or enumerate microorganisms contained in a fluid test sample.
This invention also relates to an apparatus upon which organic and/or biological molecules (or organisms) can be immobilized and reacted with reagents contained in other solutions that can be made to imbibe and/or pass through the filtration apparatus and which can be washed with solutions that can be made to imbibe and/or pass through the filtration apparatus. An example of a specific area of application is in the immobilization of a probe or probes onto the apparatus for the purpose of capturing, from a test solution, nucleic acids containing a specific nucleotide sequence represented by the complement to the nucleic acid or non-nucleic acid (e.g. Peptide Nucleic Acid) probe or probes immobilized onto the apparatus.
One area of application pertains to the microbiological testing of fluids with the goal of detecting, identifying, and/or enumerating microorganisms contained in a test fluid, or defined volume of test fluid.
Traditionally, a sample suspected of containing contaminating bacteria, yeast, or other microorganism of interest, is filtered through a sterile, microporous membrane (typically 0.22 xcexc or 0.45 xcexcporosity). The membrane is then exposed to a selective growth medium (e.g. agar or broth-based) and incubated, usually at 37xc2x0 C., until visible colonies appear on the membrane; usually 24-72 hours, depending on the species of microorganism. Incubation for a shorter time results in microscopic colonies that can very tediously be counted under a high power microscope objective. This process however, requires substantial technician time and is subject to significant counting error. When subjected to longer incubation, the visible colonies are manually counted without the use of a microscope and the original concentration of microorganisms can be calculated. Many apparati have been developed for this purpose (e.g. U.S. Pat. No. 4,317,726, U.S. Pat. No. 4,614,585, U.S. Pat. No. 4,777,137, U.S. Pat. No. 4,912,037, U.S. Pat. No. 5,202,262, U.S. Pat. No. 5,288,638, U.S. Pat. No. 4,036,698, U.S. Pat. No. 4,829,005, U.S. Pat. No. 5,409,832, U.S. Pat. No. 5,112,488, all of which are herein incorporated by reference). All of these are intended for the manual counting of colonies as described above.
A considerable amount of time can be saved if the incubation time is reduced to 4-6 hours and counting of the microscopic colonies is automated. Such a method for identification and enumeration of microorganisms has been developed and is termed Fluorescence In-Situ Hybridization (FISH) followed by automated image capture and analysis using an automated commercially available laser or charge coupled device (CCD)-based imaging system (Stender, et. al., J. Micro. Meth. 1449, 2001). This FISH method typically involves the following steps:
1. Filtration through a microporous membrane, trapping microorganisms on the membrane filter. The filter is part of a filtration apparatus with a fluid reservoir on one side and a waste receptacle on the other. Pressure can be applied to the reservoir or vacuum can be applied to the waste side in order to force the fluid through the membrane to the waste side, trapping any microorganisms on or within the filter.
2. Growth of the microorganisms on the filter by transferring the filter from the filtration apparatus to a cellulose pad or agar plug imbibed with a selective growth medium.
3. Fixation of the microorganisms on the filter with a fixative solution by transferring the filter from the growth medium to a cellulose pad imbibed with a fixative solution.
4. Hybridization on the filter with a Peptide Nucleic Acid (PNA) probe specific to the microorganism in question. The probe is labeled with a fluorescent molecule. The filter is transferred from the fixation medium to a glass slide, microorganism side up. A small amount of hybridization solution is pipetted onto the top of the membrane filter and a cover slip placed thereon in order to spread the hybridization solution over the membrane and minimize evaporation. The filter on the slide is incubated at an elevated temperature (45-65xc2x0 C.) in a humidified chamber.
5. Washing excess and unbound reagent from the filter. The slide with the filter is placed in a standard slide tray and incubated in heated wash solution. The membrane is gently teased from the slide in order to wash both sides of the membrane and remove as much hybridization solution as possible.
6. Transferring the membrane to an imager for analysis. The membrane is removed from the wash solution and placed on a microscope slide for subsequent imaging and analysis.
This process involves manually handling the delicate microporous filter directly multiple times prior to the analysis step, increasing the chance of contamination and damage.
In addition to the analysis of microorganisms or particles, this invention also pertains to microarrays. Microarrays is a fast growing field that allows for deposition of xcx9c100-200 micron spots of target molecules (in most cases, nucleic acid probes, antigens, or antibodies) that are immobilized onto a support substrate, which in most cases is a pretreated glass microscope slide. The spots are arrayed in a rectangular matrix on the surface of the slide allowing for thousands of experiments per slide. Reagents are flowed over the surface or the slides are immersed in reagent and rinse reservoirs utilizing standard equipment used for processing glass microscope slides in fields such as histochemistry, cytochemistry, immunohistochemistry, and cytogenetics. Recently, (solid) glass slides coated on one surface with a microporous matrix have become commercially available. The microporous matrix (for example, nitrocellulose, nylon) purportedly provides more surface area for immobilization and reaction with target molecules resulting in localized fluorescent, chemiluminescent, or radioactive product as an indicator that a target molecule has bound to the probe immobilized within a given xe2x80x9cspotxe2x80x9d.
U.S. Pat. No. 5,891,394 discusses an apparatus for detection and enumeration, but not identification, of microorganisms on a filter by fluorescence. Their fluorescence method is not capable of identifying a specific microorganism in the presence of other microorganisms. Furthermore, filtration and processing are done on standard 25 mm membranes. After processing, the filter is manually transferred to a well in the apparatus for analysis. There is no slide involved and the membrane is directly handled multiple times prior to analysis.
U.S. Pat. No. 4,124,449 describes a filter holder for mounting on the stage of a microscope. This apparatus allows for filtration of fluids through an integral microporous membrane, incubation of microorganisms retained on the filter with a dye to stain them, and visualization under a light or dark field microscope for the visual enumeration of microorganisms. It is not only restricted to visual observation with a microscope, but its overall dimensions far exceed those of a standard microscope slide, rendering it incompatible with standard apparatus for processing specimens on a microscope slide.
U.S. Pat. No. 5,252,293 describes a plastic disposable analytical slide with an integral microporous filter membrane. The plastic slide portion contains a number of physical features, such as slits and indentations, that allow it to align the filter in an apparatus (U.S. Pat. No. 5,529,752) designed for deposition of ligands onto specific spots on the membrane, and expose these spots to hapten-containing solutions. The slide is then moved to a measuring module within the apparatus where other features on the slide cause it to align with a measuring apparatus to quantify the amount of hapten bound. This analytical slide is for a specific application and compatible only with the apparatus of U.S. Pat. No. 5,529,752 and does not have the general applicability of the current invention, that being suitable for use in any microscope slide format.
U.S. Pat. No. 5,851,390 describes a filter membrane and housing with an elliptical shape. The elliptical shape of the assembly makes it easier to handle, particularly to attach a syringe via a luer attachment, reduces membrane waste in the manufacturing process, and facilitates addition of hydrophobic areas during the manufacturing process. The intended use is to filter entities from a fluid sample and retain the filtrate for subsequent use. The filter with its housing are discarded. The apparatus can be produced in different sizes, depending on the application, and the size of the filtration area is not restricted.
In one embodiment, the current invention pertains to a microporous filter membrane incorporated into a support having overall dimensions of a microscope slide such that the unit (Filter Slide) can be handled, viewed, stored, and otherwise processed as is any microscope slide. In another embodiment, the present invention pertains to peripheral apparati that mate with the Filter Slide providing a uniform way to filter fluids and process particles (including microorganisms) retained on the membrane of the Filter Slide.
When used with the appropriate mating apparati described herein, the current invention can be used to filter fluids through the membrane portion and retain any microscopic particles or microorganisms contained therein on one surface or trapped within the matrix of the membrane. Before and/or after observation or imaging of these entities, they can be treated with reagents that react with the retained particles or microorganisms such as to enhance their visualization. For example, the retained particles or microorganisms can be exposed to these reagents by causing the reagents to pass through the membrane with applied vacuum or pressure, or by exposing one side or the other of the membrane with a reagent and relying on diffusion to imbibe the reagent into the interstices of the membrane and thus to contact and react with the particles (including microorganisms) retained therein. These post-binding reactions usually result in a product that is fluorescent, colored, or chemiluminescent and can be imaged in any number of commercially available imaging instruments such as for example a laser based array scanner (available from suppliers such as Affymetrix, Inc., Bedford, Mass., and Axon Instruments, Inc., Foster City, Calif.) or a CCD camera equipped imaging devices (available from Applied Precision, Inc., Issaquah, Wash.)
It is an advantage of the present invention that once an image is captured, the particles or colonies of microorganisms can be counted and/or analyzed using commercially available image processing software (such as available from Media Cybernetics, Inc., Silver Spring, Md.). The further advantage is that this counting and/or analysis can be accomplished in a matter of seconds with a high degree of accuracy. Taken together these advances substantially reduce the cost and labor content of the analysis process as well as expedite the result and improve accuracy, reliability and reproducibility.
The current invention incorporates the filter membrane directly into the microscope slide dimensions. The slide, not the filter, is directly manipulated throughout any processing steps. After processing, the filter is ready for analysis without transfer to a glass slide, thus eliminating a time-consuming step and minimizing the risk of damage to the membrane. Furthermore, the invention is compatible with any visualization apparatus that accepts standard glass microscope slides and requires no additional features or special fixturing for alignment.
The current invention can be practiced with a variety of membranes, with the advantage that reagents can be brought into contact with the membrane from either side, or they can be drawn through the membrane, providing for more complete reaction and rinsing between reagent steps while still being in the format of a standard microscope slide. The flow through capability of the current invention makes it easier to automate the sample processing steps. Handling by the user is the same as with the microscope slide-based arrays they currently use.
The current invention integrates and simplifies the FISH process as described above and provides a uniform and robust way to handle and process the microorganisms retained on the filter membrane.